Abstract In type 1 diabetes (T1D) insulin producing ?-cells of the pancreatic islets of Langerhans are lost and secretion of the glucose-raising hormone glucagon from ?-cells is dysregulated, contributing to hyperglycemia and impaired counter-regulation. Recent studies demonstrate appreciable heterogeneity within the ?-cell and ?-cell populations both in vitro and in situ. Emerging single-cell approaches have established ?-cell sub-groups that differ in their Ca2+ signaling and transcriptomic profiles and may represent ?pacemaker? cells or replication niches. Evidence is also accumulating, including preliminary data in the present application, to suggest that the pancreatic ?-cells are both heterogeneous and malleable ? the altered function of human ?-cells in type 1 diabetes (T1D) is consistent with a shift towards a ?-cell phenotype. This could contribute to the dysregulation of glucagon secretion. Others have shown the persistence of ?resistant? or surviving ?-cells in T1D, both within islets and throughout the pancreas, although the nature and function of these remain unclear. Understanding the variability and malleability of human islet cell function, and the relationship of this to components of the islet microenvironment such as vasculature or nerves, is important since this may provide avenues for correction of glucagon secretory dysfunction, protection of ?-cells, or the regeneration of ?-cell mass. The present proposal will combine in-depth transcriptomic, proteomic, functional phenotyping on a cell-by-cell basis to understand the underlying regulation of islet cell functional heterogeneity and will map these in situ in relation to other islet cells types and components of the local environment. The Aims are to (1) examine human islet cell functional phenotypes, and the linkage of phenotypic variability to single-cell gene expression; (2) map the markers that define islet cell heterogeneity and sub-populations within the 3D islet microenvironment in health and T1D using approaches that span a range of resolutions and scales; and (3) link islet cell function, single-cell gene expression, single-cell metabolism, and single-cell proteomics in situ to understand islet cell pathophysiology. Integration of an in-house human islet isolation program, multi-dimensional cell imaging expertise, and single-cell dual functional and transcriptomic profiling using electrophysiology (Patch-Seq) on isolated cells and in situ using live human pancreas slices will help accomplish the goal of obtaining a high resolution understanding of islet cells within the local tissue architecture in health and diabetes.